Methods and compositions for the specific inhibition of CKAP5 by double-stranded RNA

Abstract

This invention relates to compounds, compositions, and methods useful for reducing CKAP5 target RNA and protein levels via use of dsRNAs, e.g., Dicer substrate siRNA (DsiRNA) agents.

We claim: 
     
       1. An isolated nucleic acid comprising an oligonucleotide strand of 19-50 nucleotides in length, wherein said oligonucleotide strand is complementary to SEQ ID NO: 2355 (5′-CCCAACAAGAACUAGAAGCUA-3′) along at least 19 nucleotides of said oligonucleotide strand length and reduces CKAP5 target mRNA expression when said nucleic acid is introduced into a mammalian cell. 
     
     
       2. An isolated dsNA comprising first and second nucleic acid strands, wherein said first strand is 15-35 nucleotides in length and said second strand of said dsNA is 19-50 nucleotides in length, wherein said second nucleic acid strand is complementary to SEQ ID NO: 2355 (5′-CCCAACAAGAACUAGAAGCUA-3′) along at least 19 consecutive nucleotides of said second strand length and said dsNA reduces CKAP5 target mRNA expression when said double stranded nucleic acid is introduced into a mammalian cell. 
     
     
       3. The isolated dsNA of  claim 2  comprising a duplex region selected from the group consisting of at least 25 base pairs; 19-21 base pairs and 21-25 base pairs. 
     
     
       4. The isolated dsNA of  claim 2 , wherein said second nucleic acid strand comprises 1-5 single-stranded nucleotides at its 3′ terminus. 
     
     
       5. The isolated dsNA of  claim 2 , wherein said first strand is 25-35 nucleotides in length. 
     
     
       6. The isolated dsNA of  claim 2 , wherein said second strand is 25-35 nucleotides in length. 
     
     
       7. The isolated dsNA of  claim 2 , wherein starting from the first nucleotide (position 1) at the 3′ terminus of the first strand, position 1, 2 and/or 3 is substituted with a modified nucleotide. 
     
     
       8. The isolated dsNA of  claim 2 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a blunt end. 
     
     
       9. The isolated dsNA of  claim 2 , wherein said first strand is 25 nucleotides in length and said second strand is 27 nucleotides in length. 
     
     
       10. The isolated dsNA of  claim 2 , wherein said second strand of said dsNA comprises the complement of SEQ ID NO: 2355 (5′-CCCAACAAGAACUAGAAGCUA-3′). 
     
     
       11. The isolated dsNA of  claim 2 , wherein said second strand comprises SEQ ID NO: 627 (5′-CCAAUUUAGCUUCUAGUUCUUGUUGGG-3′). 
     
     
       12. The isolated dsNA of  claim 2 , wherein said first strand comprises SEQ ID NO: 51 (5′-CAACAAGAACUAGAAGCUAAAUUgg-3′). 
     
     
       13. The isolated dsNA of  claim 2  comprising SEQ ID NO: 627 (5′-CCAAUUUAGCUUCUAGUUCUUGUUGGG-3′) and SEQ ID NO: 51 (5′-CAACAAGAACUAGAAGCUAAAUUgg-3′). 
     
     
       14. The isolated dsNA of  claim 7 , wherein said modified nucleotide residue of said 3′ terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
       15. The isolated dsNA of  claim 14 , wherein position 1 of said 3′ terminus of the first strand is a deoxyribonucleotide. 
     
     
       16. The isolated dsNA of  claim 2 , wherein said nucleotides of said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand comprise a modified nucleotide. 
     
     
       17. The isolated dsNA of  claim 16 , wherein said modified nucleotide of said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand is a 2′-O-methyl ribonucleotide. 
     
     
       18. The isolated dsNA of  claim 16 , wherein all nucleotides of said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand are modified nucleotides. 
     
     
       19. The isolated dsNA of  claim 2 , wherein said dsNA comprises a modified nucleotide. 
     
     
       20. The isolated dsNA of  claim 19 , wherein said modified nucleotide residue is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate). 
     
     
       21. The isolated dsNA of  claim 2 , wherein said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand are 1-3 nucleotides in length or are 1-2 nucleotides in length. 
     
     
       22. The isolated dsNA of  claim 2 , wherein said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand is two nucleotides in length and comprises a 2′-O-methyl modified ribonucleotide. 
     
     
       23. The isolated dsNA of  claim 2 , wherein said second strand comprises a modification pattern selected from the group consisting of AS-M1 to AS-M52 and AS-M1* to AS-M52*. 
     
     
       24. The isolated dsNA of  claim 2 , wherein said first strand comprises a modification pattern selected from the group consisting of SM1 to SM31. 
     
     
       25. The isolated dsNA of  claim 2 , wherein said dsNA is cleaved endogenously in said cell by Dicer. 
     
     
       26. The isolated nucleic acid of  claim 1 , wherein the amount of said isolated nucleic acid sufficient to reduce expression of the target gene is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of said cell. 
     
     
       27. The isolated dsNA of  claim 2 , wherein said first strand comprises SEQ ID NO: 2355 (5′-CCCAACAAGAACUAGAAGCUA 3′). 
     
     
       28. The isolated dsNA of  claim 2 , wherein said isolated dsNA is complementary to the target CKAP5 mRNA sequence to reduce CKAP5 target mRNA expression by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, and at least 99% when said double stranded nucleic acid is introduced into a mammalian cell. 
     
     
       29. The isolated dsNA of  claim 2 , wherein the first and second strands are joined by a chemical linker. 
     
     
       30. The isolated double stranded nucleic acid of  claim 2 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand are joined by a chemical linker. 
     
     
       31. The isolated double stranded nucleic acid of  claim 2 , wherein a nucleotide of said second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage. 
     
     
       32. The isolated nucleic acid of  claim 2  comprising a modification selected from the group consisting of:
 a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC) and a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, and a locked nucleic acid; 
 a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester; 
 a morpholino nucleic acid; and 
 a peptide nucleic acid (PNA). 
 
     
     
       33. A method for reducing expression of a target CKAP5 gene in a mammalian cell comprising contacting a mammalian cell in vitro with an isolated dsNA of  claim 2  in an amount sufficient to reduce expression of the target CKAP5 mRNA in said cell. 
     
     
       34. The method of  claim 33 , wherein target CKAP5 mRNA expression is reduced by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%. 
     
     
       35. The method of  claim 33 , wherein CKAP5 mRNA levels are reduced by an amount (expressed by %) of at least 90% at least 8 days after said cell is contacted with said dsNA. 
     
     
       36. The method of  claim 33 , wherein CKAP5 mRNA levels are reduced by an amount (expressed by %) of at least 70% at least 10 days after said cell is contacted with said dsNA. 
     
     
       37. A method for reducing expression of a target CKAP5 mRNA in a mammal comprising administering an isolated nucleic acid of  claim 1  to a mammal in an amount sufficient to reduce expression of a target CKAP5 mRNA in the mammal. 
     
     
       38. The method of  claim 37 , wherein said isolated nucleic acid is administered at a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said mammal per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram. 
     
     
       39. The method of  claim 37 , wherein CKAP5 mRNA levels are reduced in a tissue of said mammal by an amount (expressed by %) of at least 70% at least 3 days after said isolated dsNA is administered to said mammal. 
     
     
       40. The method of  claim 39 , wherein said tissue is selected from the group consisting of liver, pancreatic and ovarian tissues. 
     
     
       41. The method of  claim 37 , wherein said administering step comprises a mode selected from the group consisting of intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral and inhaled delivery. 
     
     
       42. A method for selectively inhibiting the growth of a cell comprising contacting a cell with an amount of an isolated nucleic acid of  claim 1  sufficient to inhibit the growth of the cell. 
     
     
       43. The method of  claim 42 , wherein said cell is a tumor cell of a subject. 
     
     
       44. The method of  claim 42 , wherein said cell is a tumor cell in vitro. 
     
     
       45. The method of  claim 42 , wherein said cell is a human cell. 
     
     
       46. A formulation comprising the isolated nucleic acid of  claim 1 , wherein said nucleic acid is present in an amount effective to reduce target CKAP5 mRNA levels when said nucleic acid is introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%. 
     
     
       47. The formulation of  claim 46 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of said cell. 
     
     
       48. A formulation comprising the isolated dsNA of  claim 2 , wherein said dsNA is present in an amount effective to reduce target CKAP5 mRNA levels when said dsNA is introduced into a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%. 
     
     
       49. The formulation of  claim 48 , wherein said effective amount is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram. 
     
     
       50. A mammalian cell containing the isolated nucleic acid of  claim 1 . 
     
     
       51. A pharmaceutical composition comprising the isolated nucleic acid of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
       52. A kit comprising the isolated nucleic acid of  claim 1  and instructions for its use. 
     
     
       53. A method for treating an CKAP5-associated disease or disorder in a subject comprising administering the isolated nucleic acid of  claim 1  and a pharmaceutically acceptable carrier to the subject in an amount sufficient to treat said CKAP5-associated disease or disorder in said subject, thereby treating of said CKAP5-associated disease or disorder in said subject. 
     
     
       54. The method of  claim 53 , wherein said CKAP5-associated disease or disorder is selected from the group consisting of renal, breast, lung, ovarian, liver, cervical, esophageal, oropharyngeal and pancreatic cancer. 
     
     
       55. A composition possessing CKAP5 inhibitory activity consisting essentially of an isolated nucleic acid of  claim 1 .